The analytic accuracy and precision for measurement of chemical analytes in vitreous humor (VH) are critical if results are to be used in forensic pathology. The purpose of our study was to demonstrate the stability and the reproducibility of VH sodium, potassium, chloride, glucose, urea nitrogen, acetone, and beta-hydroxybutyrate in specimens obtained from both eyes in medical examiner cases. We also compared with calculated VH osmolalities. Small but significant increases were observed in VH electrolyte concentrations in specimens refrigerated 6-12 months: sodium pre 144 mmol/L, post 151 mmol/L; potassium pre 12.0 mmol/L, post 12.8 mmol/L; chloride pre 121 mmol/L, post 123 mmol/L. No differences were observed between eyes, and within-day precision for all electrolyte measurements were excellent, (<1%). Frozen specimens showed significantly higher measured (439 mOsmol/kg) as compared with calculated osmolality (305 mOsmol/kg), with 1% within-day precision and no significance between eye variation for glucose and urea nitrogen. In 20 of 24 medical cases selected for possible ketoacidosis, measurement of beta-hydroxybutyrate concentrations appears to be a promising diagnostic biomarker for confirming suspected ketosis in medical examiner cases by means of VH analysis.